Method of transplanting in a mammal and treating diabetes mellitus by administering a pseudo-islet like aggregate differentiated from a nestin-positive pancreatic stem cell

ABSTRACT

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin has been identified as a molecular marker for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

This applicaion claims priority under 35 U.S.C. §119(e) to provisionalapplications, No. 60/169,082, filed on Dec. 6, 1999, Ser. No.60/215,109, filed Jun. 28, 2000 and No. 60/238,880, filed Oct. 6, 2000.

The invention was made at least in part using U.S. government funds,grants DK30457 and DK30834 awarded by the National Institutes of Health,and therefore the U.S. government may retain certain rights in theinvention.

TECHNICAL FIELD OF THE INVENTION

The invention is related to the field of stem cells and theirdifferentiation. In particular, it is related to the field of beta cellsof the islets of Langerhans in the pancreas and nestin positive liverstem cells and their differentiation from stem cells or progenitorcells, and the use of pancreatic stem cells, progenitor cells, anddifferentiated beta cells or nestin positive liver stem cells orprogenitor cells in transplantation.

BACKGROUND OF THE INVENTION

The origin of pancreatic islet cells, both during embryonic developmentand in a mature mammal, has remained uncertain despite intensive study.Certain ductal epithelial cells are capable of either differentiation ortransdifferentiation to form beta cells and other cell types found inmature islets (Bouwens, 1998). Ductal cells from isolated islets canproliferate in culture and, if transplanted into an animal, candifferentiate into functional beta cells (Cornelius et al., 1997).

It has been demonstrated that exendin-4, a long acting GLP-1 agonist,stimulates both the differentiation of β-cells from ductal progenitorcells (neogenesis) and proliferation of β-cells when administered torats. In a partial pancreatectomy rat model of type 2 diabetes, thedaily administration of exendin-4 for 10 days post pancreatectomyattenuated the development of diabetes. It has also been demonstratedthat exendin-4 stimulates the regeneration of the pancreas and expansionof β-cell mass by neogenesis and proliferation of β-cells (Xu et al.,1999, Diabetes, 48:2270-2276).

Ramiya et al. have demonstrated that islets generated in vitro frompluripotent stem cells isolated from the pancreatic ducts of adultprediabetic non-obese diabetic (NOD) mice differentiate to form glucoseresponsive islets that can reverse insulin-dependent diabetes afterbeing implanted, with or without encapsulation, into diabetic NOD mice(Ramiya et al., 2000, Nature Med., 6:278-282).

The insulinotropic hormone glucagon-like peptide (GLP)-1 which isproduced by the intestine, enhances the pancreatic expression of thehomeodomain transcription factor IDX-1 that is critical for pancreasdevelopment and the transcriptional regulation of the insulin gene.Concomitantly, GLP-1 administered to diabetic mice stimulates insulinsecretion and effectively lowers their blood sugar levels. GLP-1 alsoenhances β-cell neogenesis and islet size (Stoffers et al., 2000,Diabetes, 49:741-748).

Ferber et al. have demonstrated that adenovirus-mediated in vivotransfer of the PDX-1 (also known as IDX-1) transgene to mouse liverresults in the transconversion of a hepatocyte subpopulation towards aβ-cell phenotype. It has been demonstrated that after intravenousinfusion of mice with the PDX-1 adenoviral vector, up to 60% ofhepatocytes synthesized PDX-1. The concentration of immunoreactiveinsulin was increased in the liver and serum of treated mice. Micetreated with PDX-1 survive streptozotocin-induced diabetes, and can evennormalize glycemia (Ferber et al., 2000, Nature Med., 6:568-572).

While ductal cell cultures obtained from isolated islets apparentlycontain cells that can give rise to insulin-secreting cells, it hasremained unclear whether those cells represent true stem cells or merelyductal epithelial cells undergoing transdifferentiation. Even if suchpreparations contain genuine stem cells, it is unknown what fractionrepresent stem cells and what contaminating cell types may be present.There is a need in the art for the isolation of specific cell types frompancreatic tissue, the cell types being characterized as stem cellsusing molecular markers and demonstrated to be pluripotent and toproliferate long-term.

Pluripotent stem cells that are capable of differentiating into neuronaland glial tissues have been identified in brain. Neural stem cellsspecifically express nestin, an intermediate filament protein (Lendahlet al., 1990; Dahlstrand et al., 1992). Nestin is expressed in theneural tube of the developing rat embryo at day E11, reaches maximumlevels of expression in the cerebral cortex at day E16, and decreases inthe adult cortex, becoming restricted to a population of ependymal cells(Lendahl et al., 1990). Developing neural and pancreatic islet cellsexhibit phenotypic similarities characterized by common cellularmarkers.

The invention relates to a population of pancreatic isletstem/progenitor cells (IPCs) that are similar to neural and hepatic stemcells and differentiate into islet α-cells (glucagon) and β-cells(insulin). The invention also relates to nestin-positive liver cells.IPCs according to the invention are immunologicallysilent/immunoprivileged and are recognized by a transplant recipient asself. The IPCs according to the invention can be used for engraftmentacross allogeneic and xenogeneic barriers.

There is a need in the art for a method of engrafting stem cells acrossallogeneic and xenogeneic barriers.

There is also a need in the art for a method of treating type I diabetesmellitus wherein islets, nestin-positive pancreatic stem cells ornestin-positive liver stem cells are transferred into a recipient acrossallogeneic or xenogeneic barriers and graft rejection does not occur.

SUMMARY OF THE INVENTION

It is an object of the invention to provide mammalian pancreatic orliver stem cells for use in treating diabetes mellitus and otherdisorders. It is also an object of the invention to provide methods foridentifying, localizing, and isolating pancreatic stem cells. It is afurther object of the invention to provide methods for differentiatingpancreatic stem cells to obtain cells that produce insulin and otherhormones. It is also an object of the invention to provide methods fortransplantation into a mammal that utilize mammalian pancreatic or liverstem cells. These and other objects of the invention are provided by oneor more of the embodiments described below.

One embodiment of the invention provides a method of treating a patientwith diabetes mellitus wherein the patient does not serve as the donor.A nestin-positive pancreatic stem cell is isolated from a pancreaticislet of a donor. The stem cell is transferred into the patient, whereit differentiates into an insulin-producing cell.

Another embodiment provides another method of treating a patient withdiabetes wherein the patient does not serve as the donor. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and expanded ex vivo to produce a progenitor cell. Theprogenitor cell is transferred into the patient, where it differentiatesinto an insulin-producing beta cell. Another embodiment provides stillanother method of treating a diabetes patient wherein the patient doesnot serve as the donor. A nestin-positive pancreatic stem cell isisolated from a pancreatic islet of a donor and expanded to produce aprogenitor cell. The progenitor cell is differentiated in culture toform pseudo-islet like aggregates that are transferred into the patient.

Another embodiment provides another method of treating a patient withdiabetes mellitus wherein the patient does not serve as the donor. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and cultured ex vivo to produce a progenitor cells. Theprogenitor cell is transferred into the patient, where it differentiatesinto an insulin-producing beta cell.

In another preferred embodiment, the patient is a human and the donor isa non-human mammal.

In another preferred embodiment, the patient is not treated with animmunosuppressive agent prior to the transferring step.

In another preferred embodiment, prior to the step of transferring, thestem cell is treated ex vivo with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4,IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A,TGF-β, and combinations thereof.

In another preferred embodiment, the step of transferring is performedvia endoscopic retrograde injection.

In another preferred embodiment, the method of treating a patient withdiabetes mellitus additionally comprises the step of treating thepatient with an immunosuppressive agent.

In another preferred embodiment, the immunosuppressive agent prevents animmune response.

In another preferred embodiment, the immunosuppressive agent delays theoccurrence of an immune response.

In another preferred embodiment, the immunosuppressive agent decreasesthe intensity of an immune response.

In another preferred embodiment, the immune response is transplantrejection.

In another preferred embodiment, the immunosuppressive agent is selectedfrom the group consisting of FK-506, cyclosporin, and GAD65 antibodies.

As used herein, “differentiation” refers to the process by which a cellundergoes a change to a particular cell type, e.g. to a specialized celltype. The stem cell is treated with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleicacid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A,TGF-β, and combinations thereof. In one embodiment of the invention, thestem cell subsequently differentiates into a pancreatic progenitor cell.

In another preferred embodiment, a pancreatic progenitor subsequentlyforms pseudo-islet like aggregates.

One embodiment of the invention provides a method of transplanting intoa mammal. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor. The stem cell is transferred into themammal, where it differentiates into an insulin-producing cell.

Another embodiment provides another method of transplanting into amammal. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor and expanded ex vivo to produce a progenitorcell. The progenitor cell is transferred into the mammal, where itdifferentiates into an insulin-producing beta cell. Another embodimentprovides still another method of transplanting into a mammal. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and expanded to produce a progenitor cell. The progenitorcell is differentiated in culture to form pseudo-islet like aggregatesthat are transferred into the mammal.

Another embodiment provides another method of transplanting into amammal. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor and cultured ex vivo to produce a progenitorcells. The progenitor cell is transferred into the mammal, where itdifferentiates into an insulin-producing beta cell.

In these embodiments, the mammal can also serve as the donor of thepancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

In a preferred embodiment, the mammal does not serve as the donor of thepancreatic islet tissue.

In another preferred embodiment, the mammal is a human and the donor isa non-human mammal.

In another preferred embodiment, the mammal is not treated with animmunosuppressive agent prior to the transferring step.

In another preferred embodiment, prior to the step of transferring, thestem cell is treated ex vivo with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4,IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A,TGF-β, and combinations thereof.

In another preferred embodiment, the step of transferring is performedvia endoscopic retrograde injection.

In another preferred embodiment, the method of transplanting into amammal additionally comprises the step of treating the mammal with animmunosuppressive agent.

In another preferred embodiment, the immunosuppressive agent prevents animmune response.

In another preferred embodiment, the immunosuppressive agent delays theoccurrence of an immune response.

In another preferred embodiment, the immunosuppressive agent decreasesthe intensity of an immune response.

In another preferred embodiment, the immune response is transplantrejection.

In another preferred embodiment, the immunosuppressive agent is selectedfrom the group consisting of FK-506, cyclosporin, and GAD65 antibodies.

Yet another embodiment provides an isolated, nestin-positive humanpancreatic stem cell that is immunoprivileged. Yet another embodimentprovides an isolated, nestin-positive human pancreatic stem cell thatdoes not express class I MHC antigens. Yet another embodiment providesan isolated, nestin-positive human pancreatic stem cell that does notexpress class II MHC antigens. I Yet another embodiment provides anisolated, nestin-positive human pancreatic stem cell that does notexpress class I or class I antigens.

Yet another embodiment provides a method of treating a patient withliver disease wherein the patient does not serve as the donor. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and transferred into the patient, where the stem celldifferentiates into a hepatocyte.

In a related embodiment, the stem cell is expanded ex vivo to aprogenitor cell, which is transferred into the patient and furtherdifferentiates into a hepatocyte.

In another related embodiment, the stem cell is differentiated ex vivoto a progenitor cell, which is transferred into the patient and furtherdifferentiates into a hepatocyte. In another related embodiment, thestem cell is differentiated ex vivo into hepatocytes, which aretransplanted into the patient.

In a preferred embodiment, the patient does not serve as the donor ofthe pancreatic islet tissue.

In another preferred embodiment, the patient is a human and the donor isa non-human mammal.

In another preferred embodiment, the patient is not treated with animmunosuppressive agent prior to the transferring step.

In another preferred embodiment, the method of treating a patient withliver disease additionally comprises the step of treating the patientwith an immunosuppressive agent.

In another preferred embodiment, the immunosuppressive agent prevents animmune response.

In another preferred embodiment, the immunosuppressive agent delays theoccurrence of an immune response.

In another preferred embodiment, the immunosuppressive agent decreasesthe intensity of an immune response.

In another preferred embodiment, the immune response is transplantrejection.

Yet another embodiment provides a method of transplanting into a mammal.A nestin-positive pancreatic stem cell is isolated from a pancreaticislet of a donor and transferred into the mammal, where the stem celldifferentiates into a hepatocyte.

In a related embodiment, the stem cell is expanded ex vivo to aprogenitor cell, which is transferred into the mammal and furtherdifferentiates into a hepatocyte.

In another related embodiment, the stem cell is differentiated ex vivoto a progenitor cell, which is transferred into the mammal and furtherdifferentiates into a hepatocyte. In another related embodiment, thestem cell is differentiated ex vivo into hepatocytes, which aretransplanted into the mammal.

In these embodiments, the mammal can also serve as the donor of thepancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

In a preferred embodiment, the mammal does not serve as the donor of thepancreatic islet tissue.

In another preferred embodiment, the mammal is a human and the donor isa non-human mammal.

In another preferred embodiment, the mammal is not treated with animmunosuppressive agent prior to the transferring step.

In another preferred embodiment, the method of transplanting into amammal additionally comprises the step of treating the mammal with animmunosuppressive agent.

In another preferred embodiment, the immunosuppressive agent prevents animmune response.

In another preferred embodiment, the immunosuppressive agent delays theoccurrence of an immune response.

In another preferred embodiment, the immunosuppressive agent decreasesthe intensity of an immune response.

In another preferred embodiment, the immune response is transplantrejection.

Yet another embodiment provides an isolated, nestin-positive human liverstem cell. In versions of this embodiment, the stem cell isimmunoprivileged. In versions of this embodiment, the stem cell does notexpress class I MHC antigens. In versions of this embodiment, the stemcell does not express class II MHC antigens. In versions of thisembodiment, the stem cell does not express class I or class I antigens.

Yet another embodiment provides an isolated, nestin-positive human stemcell that is not a neural stem cell, that is capable of transplant intoan animal without causing graft versus host rejection. In versions ofthis embodiment, the stem cell is not major histocompatibility complexclass I or class II restricted.

A “stem cell” as used herein is a undifferentiated cell which is capableof essentially unlimited propagation either in vivo or ex vivo andcapable of differentiation to other cell types. This can be to certaindifferentiated, committed, immature, progenitor, or mature cell typespresent in the tissue from which it was isolated, or dramaticallydifferentiated cell types, such as for example the erythrocytes andlymphocytes that derive from a common precursor cell, or even to celltypes at any stage in a tissue completely different from the tissue fromwhich the stem cell is obtained. For example, blood stem cells maybecome brain cells or liver cells, neural stem cells can become bloodcells, such that stem cells are pluripotential, and given theappropriate signals from their environment, they can differentiate intoany tissue in the body.

In one embodiment, a “stem cell” according to the invention isimmunologically blinded or immunoprivileged. As used herein,“immunologically blinded” or “immunoprivileged” refers to a cell thatdoes not elicit an immune response. As used herein, an “immune response”refers to a response made by the immune system to a foreign substance.An immune response, as used herein, includes but is not limited totransplant or graft rejection, antibody production, inflammation, andthe response of antigen specific lymphocytes to antigen. An immuneresponse is detected, for example, by determining if transplantedmaterial has been successfully engrafted or rejected, according tomethods well-known in the art, and as defined herein in the sectionentitled, “Analysis of Graft Rejection”. In one embodiment, an“immunogically blinded stem cell” or an “immunoprivileged stem cell”according to the invention can be allografted or xenografted withouttransplant rejection, and is recognized as self in the transplantrecipient or host.

Transplanted or grafted material can be rejected by the immune system ofthe transplant recipient or host unless the host is immunotolerant tothe transplanted material or unless immunosupressive drugs are used toprevent rejection.

As used herein, a host that is “immunotolerant”, according to theinvention, fails to mount an immune response, as defined herein. In oneembodiment, a host that is “immunotolerant” does not reject or destroytransplanted material. In one embodiment, a host that is“immunotolerant” does not respond to an antigen by producing antibodiescapable of binding to the antigen.

As used herein, “rejection” refers to rejection of transplanted materialby the immune system of the host. In one embodiment, “rejection” meansan occurrence of more than 90% cell or tissue necrosis of thetransplanted material in response to the immune response of the host. Inanother embodiment, “rejection” means a decrease in the viability suchthat the viability of the transplanted material is decreased by 90% ormore as compared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.As used herein, “graft versus host rejection” or “graft versus hostresponse” refers to a cell-mediated reaction in which T-cells of thetransplanted material react with antigens of the host.

As used herein, “host versus graft rejection” or “host versus graftresponse” refers to a cell-mediated reaction in which cells of thehost's immune system attack the foreign grafted or transplantedmaterial.

In another embodiment of the invention, an immune response has occurredif production of a specific antibody (for example an antibody that bindsspecifically to an antigen on the transplanted material, or an antibodythat binds specifically to the foreign substance or a product of theforeign substance) is detected by immunological methods well-known inthe art, including but not limited to ELISA, immunostaining,immunoprecipitation and Western Blot analysis.

Stem cells express morphogenic or growth hormone receptors on the cellsurface, and can sense, for example, injury-related factors thenlocalize to and take residence at sites of tissue injury, or sense theirlocal microenvironment and differentiate into the appropriate cell type.

“Essentially unlimited propagation” can be determined, for example, bythe ability of an isolated stem cell to be propagated through at least50, preferably 100, and even up to 200 or more cell divisions in a cellculture system. Stem cells can be “totipotent,” meaning that they cangive rise to all the cells of an organism as for germ cells. Stem cellscan also be “pluripotent,” meaning that they can give rise to manydifferent cell types, but not all the cells of an organism. When a stemcell differentiates it generally gives rise to a more adult cell type,which may be a partially differentiated cell such as a progenitor cell,a differentiated cell, or a terminally differentiated cell. Stem cellscan be highly motile.

“Nestin” refers to an intermediate filament protein having a sequencedisclosed in Genbank Access No. X65964 (FIG. 7).

A “pancreatic” stem cell means a stem cell that has been isolated frompancreatic tissue and/or a cell that has all of the characteristics of:nestin-positive staining, nestin gene expression, cytokeratin-19negative staining, long-term proliferation in culture, and the abilityto differentiate into pseudo-islets in culture.

A “liver” stem cell means a stem cell that has been isolated from livertissue and/or a cell that has all of the characteristics of:nestin-positive staining, nestin gene expression, and long-termproliferation in culture.

A “progenitor cell” is a cell that is derived from a stem cell bydifferentiation and is capable of further differentiation to more maturecell types.

As used herein, the term “insulin-producing beta cell” refers to anycell which can produce and secrete insulin in a similar amount to thatproduced and secreted by a beta cell of the islets of Langerhans in thehuman pancreas. Preferably, the secretion of insulin by aninsulin-producing beta cell is also regulated in a similar fashion tothe regulation of insulin secretion by a human beta cell in situ; forexample, insulin secretion should be stimulated by an increase in theglucose concentration in the solution surrounding the insulin-producingbeta cell.

“Pseudo-islet like” aggregates are artificial aggregates ofinsulin-secreting cells which resemble in form and function the isletsof Langerhans of the pancreas. Pseudo-islet like aggregates are createdex vivo under cell culture conditions. They are approximately 50-150 μmin diameter (compared to an average diameter of 100 μm for in situislets) and spheroid in form.

“Isolating” a stem cell refers to the process of removing a stem cellfrom a tissue sample and separating away other cells which are not stemcells of the tissue. An isolated stem cell will be generally free fromcontamination by other cell types and will generally have the capabilityof propagation and differentiation to produce mature cells of the tissuefrom which it was isolated. However, when dealing with a collection ofstem cells, e.g., a culture of stem cells, it is understood that it ispractically impossible to obtain a collection of stem cells which is100% pure. Therefore, an isolated stem cell can exist in the presence ofa small fraction of other cell types which do not interfere with theutilization of the stem cell for analysis or production of other,differentiated cell types. Isolated stem cells will generally be atleast 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure.Preferably, isolated stem cells according to the invention will be atleast 98% or at least 99% pure.

A stem cell is “expanded” when it is propagated in culture and givesrise by cell division to other stem cells and/or progenitor cells.Expansion of stem cells may occur spontaneously as stem cellsproliferate in a culture or it may require certain growth conditions,such as a minimum cell density, cell confluence on the culture vesselsurface, or the addition of chemical factors such as growth factors,differentiation factors, or signaling factors.

A stem cell, progenitor cell, or differentiated cell is “transplanted”or “introduced” into a mammal when it is transferred from a culturevessel into a patient. Transplantation, as used herein, can include thesteps of isolating a stem cell according to the invention andtransferring the stem cell into a mammal or a patient. Transplantationcan involve transferring a stem cell into a mammal or a patient byinjection of a cell suspension into the mammal or patient, surgicalimplantation of a cell mass into a tissue or organ of the mammal orpatient, or perfusion of a tissue or organ with a cell suspension. Theroute of transferring the stem cell or transplantation, will bedetermined by the need for the cell to reside in a particular tissue ororgan and by the ability of the cell to find and be retained by thedesired target tissue or organ. In the case where a transplanted cell isto reside in a particular location, it can be surgically placed into atissue or organ or simply injected into the bloodstream if the cell hasthe capability to migrate to the desired target organ.

Transplantation, as used herein, can include the steps of isolating astem cell according to the invention, and culturing and transferring thestem cell into a mammal or a patient. Transplantation, as used herein,can include the steps of isolating a stem cell according to theinvention, differentiating the stem cell, and transferring the stem cellinto a mammal or a patient. Transplantation, as used herein, can includethe steps of isolating a stem cell according to the invention,differentiating and expanding the stem cell and transferring the stemcell into a mammal or a patient.

A “transplant graft” as used herein refers to at least 10⁵ stem cellsaccording to the invention and up to 10⁸ or 10⁹ stem cells.

Treatment with an immunosuppressive agent can be accomplished byadministering to a patient in need thereof any agent which prevents,delays the occurrence of or decreases the intensity of the desiredimmune response, e.g., rejection of a transplanted cell, tissue, ororgan.

As used herein, “immunosuppression” refers to prevention of the immuneresponse (for example by the administration of an “immunosuppresiveagent”, as defined herein) such that an “immune response”, as definedherein, is not detectable. As used herein, “prevention” of an immuneresponse means an immune response is not detectable. An immune response(for example, transplant rejection or antibody production) is detectedaccording to methods well-known in the art and defined herein.

“Immunosuppression” according to the invention also means a delay in theoccurrence of the immune response as compared to any one of a transplantrecipient that has not received an immunosuppresive agent, or atransplant recipient that has been transplanted with material that isnot “immunologically blinded” or “immunoprivileged”, as defined herein.A delay in the occurrence of an immune response can be a short delay,for example 1 hr-10 days, i.e., 1 hr, 2, 5 or 10 days. A delay in theoccurrence of an immune response can also be a long delay, for example,10 days-10 years (i.e., 30 days, 60 days, 90 days, 180 days, 1, 2, 5 or10 years).

“Immunosuppression” according to the invention also means a decrease inthe intensity of an immune response. According to the invention, theintensity of an immune response can be decreased such that it is 5-100%,preferably, 25-100% and most preferably 75-100% less than the intensityof the immune response of any one of a transplant recipient that has notreceived an immunosuppresive agent, or a transplant recipient that hasbeen transplanted with material that is not “immunologically blinded” or“immunoprivileged”, as defined herein. The intensity of an immuneresponse can be measured by determining the time point at whichtransplanted material is rejected. For example, an immune responsecomprising rejection of transplanted material at day 1,post-transplantation, is of a greater intensity than an immune responsecomprising the rejection of transplanted material at day 30,post-transplantation. The intensity of an immune response can also bemeasured by quantitating the amount of a particular antibody capable ofbinding to the transplanted material, wherein the level of antibodyproduction correlates directly with the intensity of the immuneresponse. Alternatively, the intensity of an immune response can bemeasured by determining the time point at which a particular antibodycapable of binding to the transplanted material is detected.

Various strategies and agents can be utilized for immunosuppression. Forexample, the proliferation and activity of lymphocytes can be inhibitedgenerally with agents such as, for example, FK-506, or cyclosporin orother immunosuppressive agents. Another possible strategy is toadminister an antibody, such as an anti-GAD65 monoclonal antibody, oranother compound which masks a surface antigen on a transplanted celland therefore renders the cell practically invisible to the immunesystem of the host.

An “immunosuppressive agent” is any agent that prevents, delays theoccurrence of or reduces the intensity of an immune reaction against aforeign cell in a host, particularly a transplanted cell. Preferred areimmunosuppressive agents which suppress cell-mediated immune responsesagainst cells identified by the immune system as non-self. Examples ofimmunosuppressive agents include but are not limited to cyclosporin,cyclophosphamide, prednisone, dexamethasone, methotrexate, azathioprine,mycophenolate, thalidomide, FK-506, systemic steroids, as well as abroad range of antibodies, receptor agonists, receptor antagonists, andother such agents as known to one skilled in the art.

A “mitogen” is any agent that stimulates mitosis and cell proliferationof a cell to which the agent is applied.

A “differentiation factor” is any agent that causes a stem cell orprogenitor cell to differentiate into another cell type. Differentiationis usually accomplished by altering the expression of one or more genesof the stem cell or progenitor cell and results in the cell altering itsstructure and function.

A “signaling factor” as used herein is an agent secreted by a cell whichhas an effect on the same or a different cells. For example, a signalingfactor can inhibit or induce the growth, proliferation, ordifferentiation of itself, neighboring cells, or cells at distantlocations in the organism. Signaling factors can, for example, transmitpositional information in a tissue, mediate pattern formation, or affectthe size, shape and function of various anatomical structures.

As used herein, a mammal refers to any mammal including but not limitedto human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cowor pig.

A “non-human mammal”, as used herein, refers to any mammal that is not ahuman.

As used herein, “allogeneic” refers to genetically different members ofthe same species.

As used herein, “isogeneic” refers to of an identical geneticconstitution.

As used herein, “xenogeneic” refers to members of a different species.

As used herein, “culturing” refers to propagating or nurturing a cell,collection of cells, tissue, or organ, by incubating for a period oftime in an environment and under conditions which support cell viabilityor propagation. Culturing can include one or more of the steps ofexpanding and proliferating a cell, collection of cells, tissue, ororgan according to the invention.

The invention also provides for a pharmaceutical composition comprisingthe isolated stem cells of the invention admixed with a physiologicallycompatible carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show dual fluorescence immunocytochemical staining ofrat pancreatic islets at embryonic day 16 (FIG. 1A) and at day 60 afterbirth (FIG. 1B). Immunostaining with an antibody for nestin is shown inwhite (red in the original, with Cy3 as fluorophore) and with anantibody for insulin is shown in grey (green in the original, with Cy2as fluorophore).

FIG. 2 shows the result of RT-PCR performed using mRNA obtained from 50rat islets. Forward and reverse primers are indicated. The depictedprimer sequences are: forward primer GCGGGGCGGTGCGTGACTAC (SEQ ID No: 3)and reverse primer GGGTGGTGAGGGTTGAGGTTTGTG (SEQ ID No: 55). The singleband of 834 bp was sequenced and identified substantially as thesequence for nestin.

FIG. 3 shows nestin-positive cells that have proliferated out from acultured rat islet.

FIG. 4 shows the development of islet like structures in culture.

FIG. 5 shows the results of RT-PCR analysis of islet-like structuresgenerated in culture. Expression of NCAM and cytokeratin-19 (CK19) wasdetected.

FIG. 6 shows the stimulation of nestin mRNA expression by high glucose.APRT was examined as a control.

FIG. 7 depicts the nestin amino acid (SEQ ID No: 2) and nucleotide (SEQID No:1) sequences.

FIGS. 8A-E depicts expression of the neural stem cell-specific markernestin in a distinct cell population within pancreatic islets asdetermined by immunocytochemistry or RT-PCR.

FIGS. 9A-C depicts characterization of nestin in stem cells isolatedfrom the pancreas by immunocytochemistry and RT-PCR.

FIGS. 10A-D depicts expression of homeodomain protein IDX-1 andproglucagon in human islet-like clusters derived from nestin-positiveislet progenitor cells (NIPs).

FIGS. 11A-C demonstrates localization of nestin-positive cells tolocalized regions of the ducts of the rat pancreas.

FIG. 12 depicts alternative models for the origin of pancreatic ductcells that are progenitors of islet endocrine cells.

FIGS. 13A and B depicts immunofluorescent staining of nestin positiveliver stem cells.

FIG. 14 depicts the sequential appearance of transcription factorsduring development of the murine endocrine pancreas.

FIGS. 15A-C depicts expression of neuroendocrine, exocrine pancreaticand hepatic markers in human NIP cultures containing stem cells.

FIG. 16 depicts expression of proglucagon and insulin mRNA as determinedby RT-PCR.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified and isolated a special subclass ofductal cells from the islets of Langerhans of mammalian pancreas thathave the functional and molecular characteristics of stem cells. Inparticular, these newly discovered pancreatic stem cells arecharacterized inter alia by one or more (and preferably all of):nestin-positive staining, nestin gene expression, cytokeratin-19negative staining, long-term proliferation in culture, and the abilityto differentiate into pseudo-islets in culture. The present inventorshave also identified liver cells that exhibit nestin-positive staining.

In one embodiment, the invention provides stem cells for a variety ofapplications, including but not limited to cellular replacement therapyfor type I insulin-dependent diabetes and other forms of diabetes aswell as the development of research tools to study the onset andprogression of various diabetic conditions, hormonal abnormalities, andgenetic diseases or conditions, such as the association of polymorphismswith particular physiologic or pathologic states. The stem cells of theinvention can also be used to carry out gene therapy of endocrinepancreatic or other tissues in isograft, allograft or xenografttransplantations. Further, the stem cells described herein can be usedto produce recombinant cells, artificial tissues, and replacement organsin culture. They can also be used for the ex vivo production of insulinand other hormones. Molecular characteristics of pancreatic stem cellsdiscovered by the inventors, such as nestin-positive and cytokeratin-19negative staining, or liver stem cells, such as nestin-positivestaining, can be used in various diagnostic, pathological, orinvestigative procedures to identify, localize, and quantitate stemcells in tissues from a patient or experimental animal.

Identification of Stem Cells in Pancreatic Islets

Previous investigators have focused on ductal epithelial cells ofpancreatic islets or exocrine tissue as a possible source of stem cellsfor the neogenesis of islet endocrine cells. Nestin is an intermediatefilament protein that was cloned by screening a cDNA library from E15rat embryos with a monoclonal antibody named R.401 (Hockfield & McKay,1985; Lendahl et al., 1990). Nestin was primarily found inneuroepithelial stem cells and is expressed in the developing centralnervous system. After maximum levels are reached in the rat embryo atE16, nestin expression declines to almost undetectable levels in adultcerebral cortex, coinciding with terminal differentiation of earlynestin-expressing progenitor cells (Lendahl et al., 1990). Nestin wasinitially found exclusively in stem cells of the embryonic developingbrain and skeletal muscle (Lendahl et al., 1990). Later studiesidentified nestin-positive neural stem cells in the subependymal layerof the adult mammalian forebrain (Morshead et al., 1994).Nestin-positive stem cells have been shown to be pluripotential evenwhen isolated from adult mice or rat brain. For example, nestin-positivestem cells can generate all three major classes of neural cells inculture: neurons, astrocytes, and oligodendrocytes (Reynolds & Weiss,1996). Nestin-positive neural stem cells respond to spinal cord injuryby proliferation and degeneration of migratory cells that differentiateinto astrocytes, participate in scar formation (Johansson et al., 1999)and restore hematopoietic cells of the bone marrow after infusion intoirradiated mice (Bjornson et al., 1999).

Characterization of Stem Cells

Stem cells according to the invention can be identified by theirexpression of nestin by, for example, FACS, immunocytochemical staining,RT-PCR, Southern Northern and Western blot analysis, and other suchtechniques of cellular identification as known to one skilled in theart.

Immunocytochemical staining, for example, is carried out according tothe following method. Cryosections (6 μM) prepared from pancreata orliver, as well as cells, are fixed with 4% paraformaldehyde inphosphate. Cells are first blocked with 3% normal donkey serum for 30min at room temperature and incubated with a primary antisera to theprotein of interest overnight at 4° C. The antisera is rinsed off withPBS and incubated with the appropriate fluorescently labeled secondaryantisera for 1 hour at room temperature. Slides are then washed with PBSand coverslipped with fluorescent mounting medium (Kirkegaard and PerryLabs, Gaithersburg, Md.). Fluorescence images are obtained using a ZeissEpifluorescence microscope equipped with an Optronics TEC-470 CCD camera(Optronics Engineering, Goleta, Calif.) interfaced with a PowerMac 7100installed with IP Lab Spectrum analysis software (Signal Analytics Corp,Vienna, Va.).

Antisera useful according to the invention include the following: mousemonoclonal antibodies to human cytokeratin 19 (clone K4.62, Sigma, St.Louis, Mo.), rabbit polyclonal antisera to rat nestin and to IDX-1(prepared by immunizations of rabbits with a purified GST-nestin fusionprotein or the last twelve amino acids of rat IDX-1, respectively)(McManus et al., 1999, J. Neurosci., 19:9004-9015), guinea piganti-insulin and anti-pancreatic polypeptide antisera, obtained fromLinco, St. Charles, Mo., and mouse antiglucagon and rabbitantisomatostatin antisera, purchased from Sigma (St. Louis, Mo.) andDAKO (Carpinteria, Calif.), respectively, mouse anti-human galanin(Peninsula Laboratories, Belmont, Calif.), collagen IV antisera (CaltagLaboratories, San Francisco, Calif.), mouse anti-rat MHC class I serum(Seroteck), and antirat MHC class II serum. The invention contemplatesthat other antisera directed to such markers is available, or will bedeveloped. Such other antisera is considered to be within the scope ofthe invention.

RT-PCR and Southern blot analysis are performed according to thefollowing methods. Total cellular RNA prepared from rat or human isletsis reverse transcribed and amplified by PCR for about 35 cyclesdepending on the desired degree of amplification, as describedpreviously (Daniel, et al., 1998, Endocrinology, 139:3721-3729).Oligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are:

-   Rat nestin: Forward 5′gcggggcggtgcgtgactac3′ (SEQ ID NO: 3);    Reverse, 5aggcaagggggaagagaaggatgt3(SEQ ID NO: 4); Hybridization,    5′aagctgaagccgaatttccttgggataccagagga3′ (SEQ ID NO: 5).-   Rat keratin 19: Forward, 5′acagccagtacttcaagacc3′(SEQ ID NO: 6);    Reverse, 5′ctgtgtcagcacgcacgtta3′(SEQ ID NO: 7); Hybridization,    5′tggattccacaccaggcattgaccatgcca3′ (SEQ ID NO: 8).-   Rat NCAM: Forward, 5′cagcgttggagagtccaaat3′(SEQ ID NO: 9); Reverse,    5′ttaaactcctgtggggttgg3′(SEQ ID NO: 10); Hybridization,    5′aaaccagcagcggatctcagtggtgtggaacgatgat3′(SEQ ID NO: 11).-   Rat IDX-1 Forward, 5′atcactggagcagggaagt3′(SEQ ID NO: 12) Reverse,    5′gctactacgtttcttatct3′ (SEQ ID NO: 13) Hybridization,    5′gcgtggaaaagccagtggg3′(SEQ ID NO: 14)-   Human nestin: Forward, 5′agaggggaattcctggag3′; (SEQ ID NO: 15)    Reverse, 5′ctgaggaccaggactctcta3′; (SEQ ID NO: 16) Hybridization,    5′tatgaacgggctggagcagtctgaggaaagt3′.(SEQ ID NO: 17)-   Human keratin: Forward, 5′cttttcgcgcgcccagcatt3′;(SEQ ID NO: 18)    Reverse, 5′gatcttcctgtccctcgagc3′;(SEQ ID NO: 19)-   Human Glut-2 Forward, 5′ gcagctgctcaactaatcac 3′(SEQ ID NO: 48)    Reverse, 5′ tcagcagcacaagtcccact 3′(SEQ ID NO: 49) Hybridization, 5′    acgggcattcttattagtcagattattggt 3′(SEQ ID NO: 50)-   Human Insulin Forward, 5′ aggcttcttctacaca3′(SEQ ID NO: 51) Reverse,    5′ caggctgcctgcacca 3′(SEQ ID NO: 52) Hybridization, 5′    aggcagaggacctgca 3′(SEQ ID NO: 53).

Other such sequences are possible and such sequences are considered tobe within the scope of the art. As a general guide, primers are selectedfrom two different exons and encompass at least one intronic sequence.In addition, an RT minus control is run for most samples. PCRamplification is effectuated at 94° C. for 1 min followed by 94° C. for10 secs, 58/56° C. for 10 secs, 72° C. for 1 min, 35 cycles, and 72° C.for 2 min. The annealing temperature is 58° C. for rat nestin and 56° C.for the remaining primer pairs.

For RT-PCR of mRNA isolated from a mammal that is not rat or human,oligonucleotides that are specific for the amplified nucleic acid fromthe mammalian species being analyzed are prepared. The selection and useof such primers is known to one skilled in the art.

For Southern hybridization oligonucleotide probes are labeled with anappropriate radionuclide, such as γ³²P ATP, using conventionaltechniques. Radiolabeled probes are hybridized to PCR productstransferred to nylon membranes at 37° C. for one hour, then washed in1×SSC+0.5% SDS at 55° C. for 10-20 min or 0.5×SCC+0.5% SDS at 42° forthe human PCR products.

Nestin as a Marker of Pancreatic Stem Cells

The inventors have now unexpectedly discovered that the pancreas ofadult mammals, including humans, contains cells that express nestin.Importantly, the distribution of nestin-positive cells in the pancreasdoes not correspond to the distribution of hormone-producing cells. Forexample, fluorescently labeled antibodies specifically reactive toinsulin or glucagon label the beta and alpha cells of the islets,respectively, whereas the inventors have observed that in mice, rats,and humans, fluorescently labeled nestin antibodies localize only tocertain cells of the ductular epithelium and not to alpha, beta, delta,and pancreatic peptide producing cells in pancreatic islets (FIG. 1).The inventors also observed that antibodies specific for collagen IV, amarker for vascular endothelial cells, galanin, a marker of nerveendings, and cytokeratin 19, a marker for ductal cells, do notcolocalize with nestin antibodies. Furthermore, the inventors have foundthat nestin-positive islet cells do not co-label with antibodies forinsulin, glucagon, somatostatin, or pancreatic polypeptide (FIG. 1).This suggests that these nestin-containing cells are not endocrinecells, ductal cells, neural cells, or vascular endothelial cells, butmay represent a truly distinct cell type within the islet that has notpreviously been described. The inventors have found nestin-positivecells in the islets, as well as localized regions of the pancreaticducts, and within centroacinar regions of the exocrine pancreas.

The expression of nestin mRNA in isolated islets was detected usingRT-PCR with RNA from isolated rat islets (FIG. 2). The functionalproperties of nestin-positive pancreatic cells were investigated usingcell culture techniques and by the isolation of nestin-positive cellsfrom islets, both of which are described below.

The inventors have also discovered that the liver of rats contains cellsthat express nestin (FIG. 13).

Cytokeratin-19 as a Marker for a Distinct Population of Duct EpithelialCells

Cytokeratin-19 (CK-19) is another intermediate filament protein. CK-19and related cytokeratins have previously been found to be expressed inpancreatic ductal cells (Bouwens et al., 1994). The inventors havediscovered, however, that while CK-19 expression is indeed confined tothe ductules, fluorescent antibodies specific for CK-19 label distinctductal cells from those labeled with nestin-specific antibodies. Thissuggests that nestin-positive cells in islets may be a distinct celltype of ductal cell from CK-19 positive cells.

Isolated Stem Cells from Pancreatic Islets and Their Use

Stem cells can be isolated from a preparation of pancreatic tissue, forexample, islets obtained from a biopsy sample of tissue from a diabeticpatient. The stem cells can then be expanded ex vivo and the resultingcells transplanted back into the donor as an isograft. Inside the donor,they may differentiate to provide insulin-secreting cells such as betacells to replace beta cells lost to the autoimmune attack which causedthe diabetes. This approach can overcome the problems of immunerejection resulting from transplantation of tissue, for example, isletsfrom another individual who might serve as the donor. In one embodimentof the invention, the use of isografted stem cells allows anothertechnique to be performed in an effort to avoid the immune rejection,namely genetic therapy of the transplanted cells to render themresistant to immune attack, such as the autoimmunity present inindividuals with type 1 diabetes. A further advantage of using stemcells over whole islets is that transplanted stem cells candifferentiate in situ and better adapt to the host environment, forexample, providing appropriate microcirculation and a complement ofdifferent islet cell types which responds to the physiological needs ofthe host. Another embodiment of the invention contemplates the use ofpartially differentiated stem cells ex vivo, for example, to formprogenitor cells, which are subsequently transplanted into the host,with further differentiation optionally taking place within the host.Although the use of an isograft of stem cells, progenitor cells, orpseudo-islets is preferred, another embodiment contemplates the use ofan allograft of stem cells, progenitor cells, or pseudo-islets obtainedfrom another individual or from a mammal of another species.

In yet another embodiment of the invention, the stem cells areimmunologically blind or immunoprivileged. In one embodiment of thisaspect of the invention, immunoprivileged stem cells do not expresssufficient amounts of class I and/or class II major histocompatibilityantigens (a.k.a. HLA or human leukocyte antigen) to elicit an immuneresponse from the host. For example, these stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

In another embodiment of this aspect of the invention, immunoprivilegedstem cells do not express class I MHC antigens and/or class II MHCantigens. These stem cells, obtained from allogeneic or xenogeneicsources do not initiate a host versus graft response in immunocompetenttransplant recipients.

In another embodiment of the invention, human tissue grafts comprisingstem cells express both human specific class I and class II MHCantigens, but are recognized by immunocompetent mice as self, and do notundergo host versus graft rejection. These stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

The invention also provides for methods of isolating stem cells from axenogenic donor, and transplanting the resulting cells into a mammal ofanother species (e.g. murine stem cells are transplanted into a human,for example, a diabetic human patient) as a xenograft.

The invention provides for methods of performing isogeneic, allogeneicor xenogeneic transplants of nestin-positive stem cells wherein the stemcells are cultured for a period of time, for example, 2-4 hours, 4-5hours, 5-10 hours or 1-3 days prior to transplantation.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of nestin-positive stem cellswherein the stem cell is expanded for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation togive rise by cell division to other stem cells or progenitor cells.

The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of stem cells wherein thenestin-positive stem cells are induced to differentiate into aprogenitor cell by treatment with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleicacid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A,TGF-β, and combinations thereof for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation. Inthe case of a pancreatic stem cell, the stem cell subsequentlydifferentiates into a pancreatic progenitor cell.

The invention provides for methods of performing isogeneic, allogeneicor xenogeneic transplants wherein nestin-positive stem cells are notcultured, expanded or differentiated prior to transplantation or whereinnestin-positive stem cells are cultured and/or expanded and/ordifferentiated prior to transplantation.

Nestin-positive cells can be proliferated in culture from isolatedpancreatic islets and subsequently isolated to form a stem cell linecapable of essentially unlimited propagation.

The inventors discovered that nestin expressing cells grow out ofcultured islets and can be observed growing around the islets as earlyas about four days in culture. These cells have a neuron-like morphology(FIG. 3), show nestin-positive staining, and express nestin mRNA. Isletscontaining nestin-positive cells can be separated from other cells,e.g., fibroblasts, that proliferate from the cultured islets by exposinga suspension of the islets to concanavalin A. The islets containingnestin-positive stem cells will not adhere to a concanavalin A coatedculture vessel, for example, allowing the islets to be simply decantedwhile other cell types remain attached to the vessel. The islets arethen plated on wells that do not have a concanavalin A coating, wherethey adhere. The details of the culture and isolation procedure aredescribed for rat cells in Example 1 below. Similar results have beenobtained with human cells.

Formation of Pseudo-Islets and Ductal Structures in Culture

One embodiment of the invention provides an alternative totransplantation of stem cells or progenitor cells by causing them toform pseudo-islet like aggregates that can be transplanted into apatient with insufficient islet cell mass to maintain physiologicalcontrol without hormone therapy. Islet-derived stem cells can beprepared from cultured islets as indicated above or obtained from apropagating stem cell line. The stem cells can then be induced todifferentiate by exposing them to various growth factors. This processis illustrated in Examples 2 and 3.

Differentiation of Stem Cells or Progenitor Cells to Islet Cells

Growth factors that may induce differentiation of pancreatic stem cellsinclude but are not limited to EGF-2, basic FGF, high glucose, KGF,HGF/SF, GLP-1, exendin-4, betacellulin, activin A, TGF-β, andcombinations thereof. GLP-1 refers to glucagon-like peptide-1. Highglucose refers to a higher glucose concentration than the concentrationnormally used in culturing the stem cells. For example, the stem cellscan be normally cultured and propagated in about 5.6 mM glucose, and thehigh glucose concentration refers to another concentration above 5.6 mM.In the preferred embodiment, a concentration of 16.7 mM is contemplated.In Example 2, one possible growth factor treatment is described usingbasic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

In addition to growth factors added to the medium of cultured cells,further growth factors can contribute to differentiation when stem cellsare implanted into an animal or a human. In that situation, many growthfactors which are either known or unknown may be secreted by endogenouscells and exposed to the stem cells in situ. Implanted stem cells can beinduced to differentiate by any combination of endogenous andexogenously administered growth factors that are compatible with thedesired outcome, i.e., the final differentiated cell type or types andthe anatomical structures (e.g., tissues or organs) formed.

One embodiment provides an approach to stimulating differentiation, thatis to administer downstream effectors of growth factors or to transfectstem cells or progenitor cells with a nucleic acid molecule encodingsuch effectors. One example is IDX-1, which is a transcription factorinduced by GLP-1 or exendin-4. Introducing effectors such as IDX-1 cantrigger differentiation to form endocrine islet cells.

Analysis of Graft Rejection

The invention provides for an in vivo procedure for evaluating thesurvival of transplanted material. Experimental transplant rejection isanalyzed by transplanting an immunosuppressed or a non-immunosuppressedmammal, with a stem cell or a pseudo-islet like aggregate according tothe invention.

For example, non-immunosuppressed C57BL/6 mice are transplanted (forexample, under the renal capsules) with human stem cells according tothe invention. Graft rejection is analyzed by sacrificing the transplantrecipient and staining for viability, or performing immunocytochemicalstaining at the site of the grafted material (i.e., an organ or tissuepresent at the site of the grafted material) at a suitablepost-transplantation time point. The time point at which staining (forexample hematoxylin/eosin or immunostaining) of the site of the graftedmaterial is made can vary, for example, according to the averagesurvival time, or the expected survival time of a transplanted mammal.The site of the graft is analyzed, for example by staining, 1 day to 10years (i.e., 1, 5, 10, 30, 100 or more days, 1, 2, 5, or 10 years)post-transplantation, preferably 10 days to 1 year post-transplantationand most preferably, 10-100 days post-transplantation. For example, iftransplanted material is introduced under the renal capsule of a mouse,the kidney of the transplanted mouse is inspected. Transplanted materialis successfully engrafted (i.e., not rejected) if, the transplantedmaterial is still detectable and/or the transplanted material hasproliferated into a tissue mass.

Detection of the transplanted material and proliferation of thetransplanted material is determined, for example, by hematoxylin/eosinstaining of a frozen section prepared from the transplant site (i.e.,the kidney) and the detection of new growth that is not derived from thetransplant recipient (i.e., not host kidney derived). In the case ofxenogeneic transplantation, transplanted material is successfullyengrafted if specific immunostaining with antisera specific for anantigen from the species from which the transplanted material isderived, according to methods of immunocytochemical staining known inthe art and described herein, identifies positive cells. Alternatively,in embodiments wherein a xenogeneic transplantation is performed,transplanted material is successfully engrafted if molecules (i.e., aprotein or an antigen) derived from the transplant species (that is thespecies from which the transplanted material is derived) are detected inthe blood of the transplant recipient.

As used herein, “rejection” refers to rejection of transplanted materialby the immune system of the host. In one embodiment, “rejection means anoccurrence of more than 90% cell or tissue necrosis of the transplantedmaterial in response to the immune response of the host. In anotherembodiment, “rejection” means a decrease in the viability such that theviability of the transplanted material is decreased by 90% or more ascompared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.

Methods of Transplantation

The invention provides for methods of transplantation in to a mammal. Astem cell, progenitor cell, or differentiated cell is “transplanted” or“introduced” into a mammal when it is transferred from a culture vesselinto a patient.

Transplantation, according to the invention can include the steps ofisolating a stem cell according to the invention and transferring thestem cell into a mammal or a patient. Transplantation according to theinvention can involve transferring a stem cell into a mammal or apatient by injection of a cell suspension into the mammal or patient,surgical implantation of a cell mass into a tissue or organ of themammal or patient, or perfusion of a tissue or organ with a cellsuspension. The route of transferring the stem cell or transplantation,will be determined by the need for the cell to reside in a particulartissue or organ and by the ability of the cell to find and be retainedby the desired target tissue or organ. In the case where a transplantedcell is to reside in a particular location, it can be surgically placedinto a tissue or organ or simply injected into the bloodstream if thecell has the capability to migrate to the desired target organ.

Transplantation, according to the invention, can include the steps ofisolating a stem cell according to the invention, and culturing andtransferring the stem cell into a mammal or a patient. In anotherembodiment, transplantation, as used herein, can include the steps ofisolating a stem cell according to the invention, differentiating thestem cell, and transferring the stem cell into a mammal or a patient.Transplantation, as used herein, can include the steps of isolating astem cell according to the invention, differentiating and expanding thestem cell and transferring the stem cell into a mammal or a patient.

Methods of Treating Insulin-Dependent Diabetes Using Pancreatic StemCells

Stem cells are useful to replace lost beta cells from Type 1 diabetespatients or to increase the overall numbers of beta cells in Type 2diabetes patients. The diabetes patient will preferably serve as thedonor of pancreatic tissue used to produce stem cells, progenitor cells,or pseudo-islet like aggregates. Stem cells exist within the adultpancreatic islets as well as the pancreatic ducts. After a diabeticpatient undergoes pancreatic biopsy, islets are isolated from the biopsytissue and prepared for culture ex vivo preferably within 24 hours. Stemcells can be proliferated and isolated by the methods described abovewithin 2-3 weeks. Stem cells can be transplanted back into the patientdirectly following isolation or after a period of differentiation whichis induced by growth factors. Islets can be produced by subculture asdescribed in Example 2. The whole process of surgical pancreas biopsyand transplantation can be performed within a period of about 30 days.

In one embodiment of the invention, pluripotential stem cells are used.These cells are immunologically blinded or immunoloprivileged, such thatin allogeneic or xenogeneic transplants, they are recognized as self bythe recipient, and are not MHC restricted by class I or class IIantigens. In one aspect of this embodiment of the invention, these cellsdo not express MHC class I and/or class II antigens.

In another embodiment of the invention, the recipient of the transplantmay demonstrate host vs. graft rejection of other transplanted cells,which can be combated by the administration of blocking antibodies to,for example, an autoantigen such as GAD65, by the administration of oneor more immunosuppressive drugs described herein, or by any method knownin the art to prevent or ameliorate autoimmune rejection.

Alternatively, stem cells isolated from a non-human mammal according tothe invention, are transplanted into a human diabetes patient. Prior tothe transplantation step the stem cells may be cultured, and/or expandedand/or differentiated.

Methods of Treating Patients Suffering from Liver Disease UsingPancreatic Stem Cells

The ability of pancreatic stem or progenitor cells to transdifferentiateto form hepatocytes is well known (Bisgaard & Thorgeirsson, 1991). Thepancreatic stem cells of the present invention can be used to providehepatocytes for a patient suffering from a liver disease such ascirrosis, hepatitis, or hepatic cancer in which the functional mass ofhepatic tissue has been reduced. The stem cells of the invention canalso be treated by gene therapy to correct a genetic defect andintroduced into a patient to restore hepatic function. Nestin-positivestem cells can be differentiated either in culture or in vivo byapplying one or more growth factors, or other treatment such astransfection with a nucleic acid molecule, that results indifferentiation of the stem cells to hepatocytes. In one embodiment, theinvention contemplates the use of cyclopamine to suppress, for example,sonic hedgehog, resulting in hepatocyte formation. In another embodimentof the invention, the stem cells can be transplanted without any ex vivotreatment and the appropriate growth factors can be provided in situwithin the patient's body. In yet another embodiment, the stem calls canbe treated with growth factors or other agents ex vivo and subsequentlytransplanted into the patient in a partially differentiated orterminally differentiated state. Other aspects of the invention,including methods of transfecting stem cells or progenitor cells,dosages and routes of administration, pharmaceutical compositions,donor-isograft protocols, and immunosuppression methods, can bepracticed with transdifferentiation to hepatocytes just as for thedifferentiation to pancreatic tissues.

The invention specifically contemplates transplanting into patientsisogeneic, allogeneic, or xenogeneic stem cells, or any combinationthereof.

Methods of Stem Cell Transfection

A variety of methods are available for gene transfer into pancreaticstem cells. Calcium phosphate precipitated DNA has been used butprovides a low efficiency of transformation, especially for nonadherentcells. In addition, calcium phosphate precipitated DNA methods oftenresult in insertion of multiple tandem repeats, increasing thelikelihood of disrupting gene function of either endogenous or exogenousDNA (Boggs, 1990). The use of cationic lipids, e.g., in the form ofliposomes, is also an effective method of packaging DNA for transfectingeukaryotic cells, and several commercial preparations of cationic lipidsare available. Electroporation provides improved transformationefficiency over the calcium phosphate protocol. It has the advantage ofproviding a single copy insert at a single site in the genome. Directmicroinjection of DNA into the nucleus of cells is yet another method ofgene transfer. It has been shown to provide efficiencies of nearly 100%for short-term transfection, and 20% for stable DNA integration.Microinjection bypasses the sometimes problematic cellular transport ofexogenous DNA through the cytoplasm. The protocol requires small volumesof materials. It allows for the introduction of known amounts of DNA percell. The ability to obtain a virtually pure population of stem cellswould improve the feasibility of the microinjection approach to targetedgene modification of pancreatic stem cells. Microinjection is a tedious,highly specialized protocol, however. The very nature of the protocollimits the number of cells that can be injected at any given time,making its use in large scale production limited. Gene insertion intopancreatic stem cells using retroviral methods is the preferred method.Retroviruses provide a random, single-copy, single-site insert at veryhigh transfection efficiencies. Other such transfection methods areknown to one skilled in the art and are considered to be within thescope of this invention.

Retroviral Transformation of Pancreatic Stem Cells

Gene transfer protocols for pancreatic cells can involve retroviralvectors as the “helper virus” (i.e., encapsidation-defective viralgenomes which carry the foreign gene of interest but is unable to formcomplete viral particles). Other carriers such as DNA-mediated transfer,adenovirus, SV40, adeno-associated virus, and herpes simplex virusvectors can also be employed. Several factors should be considered whenselecting the appropriate vector for infection. It is sometimespreferable to use a viral long terminal repeat or a strong internalpromoter to express the foreign gene rather than rely on splicedsubgenomic RNA.

The two primary methods of stem cell transformation are co-culture andsupernatant infection. Supernatant infection involves repeated exposureof stem cells to the viral supernatant. Co-culture involves thecommingling of stem cells and an infected “package cell line” (seebelow) for periods of 24 to 48 hours. Co-culture is typically moreefficient than supernatant infection for stem cell transformation. Afterco-culture, infected stem cells are often further cultured to establisha long term culture (LTC).

The cell line containing the helper virus is referred to as the packagecell line. A variety of package cell lines are currently available. Animportant feature of the package cell line is that it does not producereplication-competent helper virus.

In one embodiment of the invention animals or patients from whom stemcells are harvested may be treated with 5-fluorouracil (5-FU) prior toextraction. 5-FU treated stem cells are more susceptible to retroviralinfection than untreated cells. 5-FU stem cells dramatically reduce thenumber of clonogenic progenitors, however.

In another embodiment, harvested stem cells may be exposed to variousgrowth factors, such as those employed to promote proliferation ordifferentiation of pancreatic stem cells. Growth factors can beintroduced in culture before, during, or after infection to improve cellreplication and transduction. Studies report the use of growth factorsincrease transformation efficiency from 30 to 80%.

Typical Retroviral Transformation Protocol

The ex vivo transduction of mammalian pancreatic stem cells andsubsequent transplantation into nonablated recipients sufficient toobtain significant engraftment and gene expression in various tissuescontaining their progeny cells has been shown in mice. The target cellsare cultured for 2-4 days in the presence of a suitable vectorcontaining the gene of interest, before being injected in to therecipient.

Specifically, bone marrow stem cells were harvested from male donor (4-8weeks old) BALB/c AnNCr mice (National Cancer Institute, Division ofCancer Treatment Animal Program, Frederick, Md.). The cells were platedat a density of 1-2×10⁷ cells/10 cm dish and cultured for 48 hours inDulbecco's modified Eagle's medium (DMEM) containing; 10%heat-inactivated fetal bovine serum, glutamine, Pen/Strep, 100 U/ml ofinterleukin-6 (IL-6) and stem cell factor (SCF; Immunex, Seattle, Wash.)to stimulate cell growth (Schiffmann, et. al., 1995).

Concurrently, a viral package cell line was cultured for 24 hours. Thepackage cell line used by Schiffinann, et al. was GP+E86 and the viralvector was the LG retroviral vector based on the LN series of retroviralvectors.

After the appropriate incubation period, 1-2×10⁷ stem cells were platedon a 10 cm dish containing the viral package cells and co-cultured for48 hours in the presence of 8 μg/ml of polybrene and under the samegrowth factor stimulation conditions as the donor stem cells. The stemcells were then harvested, washed of growth media and injected intorecipient mice at dosages of 2×10⁷ cells/injection for multipleinjections (total of 5 injections either daily or weekly).

Successful stem cell transduction and engraftment of stem cells can bedetermined through, for example, PCR analysis, immunocytochemicalstaining, Southern Northern or Western blotting, or by other suchtechniques known to one skilled in the art.

Mammals

Mammals that are useful according to the invention include any mammal(for example, human, mouse, rat, sheep, rabbit, goat, monkey, horse,hamster, pig or cow). A non-human mammal according to the invention isany mammal that is not a human, including but not limited to a mouse,rat, sheep, rabbit, goat, monkey, horse, hamster, pig or a cow.

Dosage and Mode of Administration

By way of example, a patient in need of pancreatic stem cells asdescribed herein can be treated as follows. Cells of the invention canbe administered to the patient, preferably in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle, byingestion, injection, inhalation or any number of other methods. Apreferred method is endoscopic retrograde injection. Another preferredmethod is injection or placement of the cells or pseudo-islet likeaggregates into the space under the renal capsule. The dosagesadministered will vary from patient to patient; a “therapeuticallyeffective dose” can be determined, for example but not limited to, bythe level of enhancement of function (e.g., insulin production or plasmaglucose levels). Monitoring levels of stem cell introduction, the levelof expression of certain genes affected by such transfer, and/or thepresence or levels of the encoded product will also enable one skilledin the art to select and adjust the dosages administered. Generally, acomposition including stem cells will be administered in a single dosein the range of 10⁵-10⁸ cells per kg body weight, preferably in therange of 10⁶-10⁷ cells per kg body weight. This dosage may be repeateddaily, weekly, monthly, yearly, or as considered appropriate by thetreating physician. The invention provides that cell populations canalso be removed from the patient or otherwise provided, expanded exvivo, transduced with a plasmid containing a therapeutic gene ifdesired, and then reintroduced into the patient.

Pharmaceutical Compositions

The invention provides for compositions comprising a stem cell accordingto the invention admixed with a physiologically compatible carrier. Asused herein, “physiologically compatible carrier” refers to aphysiologically acceptable diluent such as water, phosphate bufferedsaline, or saline, and further may include an adjuvant. Adjuvants suchas incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,or alum are materials well known in the art.

The invention also provides for pharmaceutical compositions. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carrier preparations which can beused pharmaceutically.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer' solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For nasal administration, penetrants appropriate to the particularbarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a Ph range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition with information including amount, frequency andmethod of administration.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples, which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLE 1

Isolation of Nestin-Positive Stem Cells from Rat Pancreas

Rat islets were isolated from the pancreata of 2-3 month oldSprague-Dawley rats using the collagenase digestion method described byLacy and Kostianovsky. Human islets were provided by the DiabetesResearch Institute, Miami, Fla. using collagenase digestion. The isletswere cultured for 96 hrs at 37° C. in 12-well plates (Falcon 3043plates, Becton Dickinson, Lincoln Park, N.J.) that had been coated withconcanavalin A. The culture medium was RPMI 1640 supplemented with 10%fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, 100 μg/mlstreptomycin, 100 units/ml penicillin, 0.25 μg/ml amphotericin B (GIBCOBRL, Life Science Technology, Gaithersburg, Md.), and 71.5 mMβ-mercaptoethanol (Sigma, St. Louis, Mo.).

After 96 hrs, fibroblasts and other non-islet cells had adhered to thesurface of concanavalin A coated wells and the islets remained floating(did not adhere to the surface). At this time, the media containing theislets were removed, centrifuged down, and the purged islets replated in12-well plates without a coating of concanavalin A. The islets were thencultured in the above RPMI 1640 medium supplemented with 20 ng/ml ofbasic fibroblast growth factor-2 and 20 ng/ml of epidermal growthfactor.

The islets adhered to the surface of the plates, and cells grew out andaway from the islets in a monolayer. These cells that form a monolayerwere nestin-positive by immunostaining with a rabbit anti-rat nestinantiserum developed by Dr. Mario Vallejo at the Massachusetts GeneralHospital. Other nestin antibodies may be used, for example the R.401antibody described hereinabove, or the MAB533 antibody. A monoclonalantibody specific for rat embryo spinal cord nestin, MAB353, ATCC No.1023889; is described in Journal of Neuroscience 1996; 16:1901-100; andalso available from Chemicon International, Single Oak Dr., Temecula,Calif. 92590 USA. After two weeks of culture, several (3-5) of thenestin-positive monolayer cells were removed by picking with a capillarytube (cylinder cloning) and were replated on the 12-well plates (notcoated with concanavalin A) and cultured in the RPMI 1640 medium furthersupplemented with bFGF-2 and EGF. The cells propagated at a rapid rateand reached confluence after six days of culture. After 12 days ofculture, the cell monolayer formed waves in which they begin to pile upin a co-linear manner. On day 15 of culture, the cell waves began tocondense, migrate into spheroid bodies and by day 17 the surface of thewells contained these spheroid bodies (ca. 100 μm in diameter), emptyspaces, and a few areas of remaining monolayer cells. Several of thesemonolayer cells were re-picked and re-cloned and the process describedabove occurred again in precisely the same temporal sequence.

EXAMPLE 2

Differentiation of Pancreatic Stem Cells to Form Islet

Pancreatic islets from rats were first cultured in RPMI mediumcontaining 10% fetal bovine serum using concanavalin-A coated 12-wellplates. The islets were maintained in culture for three days in theabsence of added growth factors other than those supplied by fetalbovine serum. After this period, during which the islets did not attach,the islets were transferred to fresh plates without concanavalin A. Thestem cells were then stimulated to proliferate out from the islets as amonolayer by exposing them to bFGF-2 (20 ng/ml) and EGF (20 ng/ml) for24 days. After the 24 day period, the monolayer was confluent. Amongthem was a population of cells surrounding the islets. Cells from thatpopulation were picked and subcloned into new 12-well plates and againcultured in the medium containing bFGF and EGF. The subcloned cellsproliferated rapidly into a monolayer in a clonal fashion, expandingfrom the center to the periphery. The cells became confluent at day 6and then started to form a wave of overlapping cells on day 12. By day17 the cells migrated almost entirely into spherical structures andtubular structures resembling islet-like structures (pseudo-islet likeaggregates) and duct-like structures (pseudo-ducts) (FIG. 4). RT-PCRanalysis revealed that the pseudo-islet like aggregates were expressingNCAM (a marker for endocrine cells, see FIG. 5), cytokeratin 19 (amarker for ductal cells, see FIG. 5), and the transcription factorbrain-4 (a beta cell marker). Treatment with growth factors is requiredto achieve terminal differentiation to mature islet cells.

EXAMPLE 3

Isolation and Culture of Human or Rat Pancreatic Islets

Human pancreatic islets were isolated and cultured. Human islet tissuewas obtained from the islet distribution program of the Cell TransplantCenter, Diabetes Research Institute, University of Miami School ofMedicine and the Juvenile Diabetes Foundation Center for IsletTransplantation, Harvard Medical School, Boston, Mass. Thoroughly washedislets were handpicked, suspended in modified RPMI 1640 media (11.1 mMglucose) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 1mM sodium pyruvate, 100 U per mL penicillin G sodium, 100 μg per mLstreptomycin sulfate, 0.25 ng per mL amphotericin B, and 71.5 μMβ-mercaptoethanol, and added to Falcon 3043 12-well tissue cultureplates that had been coated with Concanavalin A (ConA). The isletpreparation was incubated for 96 hrs at 37° C. with 95% air and 5% CO₂.In these conditions, many islets remained in suspension (floated),whereas fibroblasts and other non-islet cells attached to thesubstratum. After 96 h of incubation the media containing the suspendedislets was carefully removed, the islets were manually picked andresuspended in the modified RPMI 1640 media now further supplementedwith 20 ng/mL each of basic fibroblast growth factor (bFGF) andepidermal growth factor (EGF). The islet suspension (containing 20-30islets per well) was added to 12-well tissue culture plates not coatedwith ConA. The islets immediately adhered to the surfaces of the plates.Within several days, a monolayer of cells was observed growing out andaway from the islets. In certain instances, human-derived cells werecultured in modified RPMI media containing 2.5 mM glucose, and severalgrowth factor combinations that include activin-A (2 nM), hepatocytegrowth factor (100 pM), or betacellulin (500 pM). In those experimentsin which cells were challenged with 10 mM nicotinamide, the mediacontained no serum or growth factors.

EXAMPLE 4

Effects of Glucose and GLP-1 on Differentiation of Pancreatic Stem Cells

Elevation of plasma glucose concentration leads to increased pancreaticislet size. The effect of the glucose concentration in the culturemedium was therefore investigated using isolated islets, which containnestin-positive stem cells. Rat pancreatic islets were cultured in amedium containing high (16.7 mM) glucose or in normal (5.6 mM) glucose.After four days, RT-PCR was performed to determine the level of nestinmRNA. The results indicated a three-fold stimulation of nestin mRNAlevels in the islets cultured in high glucose compared to the isletscultured in normal glucose (FIG. 6).

Similarly, injection of glucagon-like peptide-1 (GLP-1) into mice wasfound to increase islet mass by 2-fold in 48 hours. Knockout mice havinga disrupted gene for GLP-1 receptor were examined for nestin expressionin pancreatic islets. Immunostaining using a nexin antibody was found tobe markedly reduced compared to normal mice with GLP-1 receptors.

Animal Model of Diabetes Mellitus

Treatments for diabetes mellitus type that result in relief of itssymptoms are tested in an animal which exhibits symptoms of diabetes. Itis contemplated that the animal will serve as a model for agents andprocedures useful in treating diabetes in humans. Potential treatmentsfor diabetes can therefore be first examined in the animal model byadministering the potential treatment to the animal and observing theeffects, and comparing the treated animals to untreated controls.

The non-obese diabetic (NOD) mouse is an important model of type I orinsulin dependent diabetes mellitus and is a particularly relevant modelfor human diabetes (see Kikutano and Makino, 1992, Adv. Immunol. 52:285and references cited therein, herein incorporated by reference). Thedevelopment of type I diabetes in NOD mice occurs spontaneously andsuddenly without any external stimuli. As NOD mice develop diabetes,they undergo a progressive destruction of β-cells which is caused by achronic autoimmune disease. The development of insulin-dependentdiabetes mellitus in NOD mice can be divided roughly into two phases:initiation of autoimmune insulitis (lymphocytic inflammation in thepancreatic islets) and promotion of islet destruction and overtdiabetes. Diabetic NOD mice begin life with euglycemia, or normal bloodglucose levels, but by about 15 to 16 weeks of age the NOD mice startbecoming hyperglycemic, indicating the destruction of the majority oftheir pancreatic β-cells and the corresponding inability of the pancreasto produce sufficient insulin. In addition to insulin deficiency andhyperglycemia, diabetic NOD mice experience severe glycosuria,polydypsia, and polyuria, accompanied by a rapid weight loss. Thus, boththe cause and the progression of the disease are similar to humanpatients afflicted with insulin dependent diabetes mellitus. Spontaneousremission is rarely observed in NOD mice, and these diabetic animals diewithin 1 to 2 months after the onset of diabetes unless they receiveinsulin therapy.

The NOD mouse is used as an animal model to test the effectiveness ofthe various methods of treatment of diabetes by administering a stemcell preparation according to the invention. As such, treatment viaadministration of stem cells are tested in the NOD mouse for theireffect on type I diabetes.

The stem cells are administered to a NOD mouse, typicallyintraperitoneally, according to the following dosage amounts. NOD miceare administered about 1×10¹ to 1×10⁴ cells per mouse. Administration ofthe cells is started in the NOD mice at about 4 weeks of age, and iscontinued for 8 to 10 weeks, e.g., 3 times a week. The mice aremonitored for diabetes beginning at about 13 weeks of age, being testedtwice per week according to the methods described below. The effects oftreatment are determined by comparison of treated and untreated NODmice.

The effectiveness of the treatment methods of the invention on diabetesin the NOD mice is monitored by assaying for diabetes in the NOD mice bymeans known to those of skill in the art, for example, examining the NODmice for polydipsia, polyuria, glycosuria, hyperglycemia, and insulindeficiency, or weight loss. For instance, the level of urine glucose(glycosuria) can be monitored with Testape (Eli Lilly, Indianapolis,Ind.) and plasma glucose levels can be monitored with a Glucometer 3Blood Glucose Meter (Miles, Inc., Elkhart, Ind.) as described by Burkly,1999, U.S. Pat. No. 5,888,507, herein incorporated by reference.Monitoring urine glucose and plasma glucose levels by these methods, NODmice are considered diabetic after two consecutive urine positive testsgave Testape values of +1 or higher or plasma glucose levels>250 mg/dL(Burkly, 1999, supra). Another means of assaying diabetes in NOD mice isto examine pancreatic insulin levels in NOD mice. For example,pancreatic insulin levels can be examined by immunoassay and comparedamong treated and control mice (Yoon, U.S. Pat. No. 5,470,873, hereinincorporated by reference). In this case, insulin is extracted frommouse pancreas and its concentration is determined by itsimmunoreactivity, such as by radioimmunoassay techniques, using mouseinsulin as a standard.

In addition to monitoring NOD mice for diabetes in general, the effectsof the inventive methods of treatment are also monitored forgene-specific or gene product-specific effects if the stem cellsadministered were transformed or transfected with a heterologous gene,thereby allowing a correlation to be drawn between expression of theheterologous gene and its effects on diabetes. For example, the presenceof the heterologous gene product may be examined by immunohistochemistryof the pancreatic β-cells of NOD mice for the gene product and forinsulin. The expression of the patched and smoothened genes is furtherexamined in NOD mouse islets by detection of the RNA transcript for thepatched and smoothened receptors. Reverse transcription-polymerase chainreaction (RT-PCR) amplification is performed by known means to amplify afragment of mouse patched or smoothened cDNA, and analyzed by agarosegel electrophoresis, according to standard means. The identification ofthe amplified cDNA fragment is confirmed as corresponding to the patchedor smoothened RNA by hybridization of the amplified fragment with aradiolabeled internal oligonucleotide probe for the patched orsmoothened genes, or by other such methods as known to one skilled inthe art.

EXAMPLE 5

Immunocytochemical Identification of Nestin Positive Human and RatPancreatic Stem Cells

Pancreatic islets were analyzed for nestin expression. Islets and stemcells were isolated as described above. Nestin expression was observedby immunocytochemical staining in a distinct population of cells withindeveloping islet clusters of embryonic day 16 (E16) rat pancreas (FIG.8A) and in islets of the adult pancreas (postnatal 60 days) (FIG. 8B).Immunocytochemical staining was performed as follows.

Cryosections (6 μM) prepared from embryonic day 16 and adult (60 day)rat pancreata as well as cells were fixed with 4% paraformaldehyde inphosphate. Cells were first blocked with 3% normal donkey serum for 30min at room temperature and incubated with primary antisera overnight at4° C. The antisera were rinsed off with PBS and incubated with therespective Cy-3 and Cy-2 labeled secondary antisera for 1 hour at roomtemperature. Slides were then washed with PBS and coverslipped withfluorescent mounting medium (Kirkegaard and Perry Labs, Gaithersburg,Md.). Tissue sections were incubated overnight at 4° C. with primaryantisera. Primary antisera were then rinsed off with PBS, and slideswere blocked with 3% normal donkey serum for 10 min at room temperaturebefore incubation with donkey anti-Cy3 (indocarbocyanine) and eitheranti-guinea pig (insulin), anti-mouse (glucagon), or anti-sheep(somatostatin) sera DTAF (Jackson Immuno Research Laboratories, WestGrove, Pa.) for 30 min at room temperature. Slides were then rinsed withPBS and coverslipped with fluorescent mounting medium (Kirkegaard andPerry Laboratories, Gaithersburg, Md.). Fluorescence images wereobtained using a Zeiss Epifluorescence microscope equipped with anOptronics TEC-470 CCD camera (Optronics Engineering, Goleta, Calif.)interfaced with a PowerMac 7100 installed with IP Lab Spectrum analysissoftware (Signal Analytics Corp, Vienna, Va.).

The nestin-positive cells are distinct from β-, α-, δ-, and PP-cellsbecause they do not co-stain with antisera to the hormones insulin(FIGS. 8A & B), glucagon, somatostatin, or pancreatic polypeptide. Thenestin-positive cells also do not co-stain with antisera to collagen IV,a marker for vascular endothelial cells (FIG. 8C) nor with an antiserumto galanin, a marker for nerve cells or a monoclonal antibody tocytokeratin 19, a specific marker for ductal cells (FIG. 8).Nestin-positive staining is associated with distinct cells within theislets clearly observed by nuclear costaining (FIG. 4D).

EXAMPLE 6

Identification of Nestin Positive Human and Rat Stem Cells by RT-PCR

To confirm the immunocytochemical identification of nestin expression inpancreatic islets, we performed an RT-PCR of the nestin mRNA using totalRNA prepared from freshly isolated rat islets and human islet tissue.RT-PCR was performed according to the following method.

Total cellular RNA prepared from rat or human islets was reversetranscribed and amplified by PCR for 35 cycles as described previously(Daniel et al., 1998, Endocrinology, 139:3721-3729). Theoligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are:

-   Rat nestin: Forward, 5′gcggggcggtgcgtgactac3′ (SEQ ID NO: 3);    Reverse, 5′aggcaagggggaagagaaggatgt3′(SEQ ID NO: 4); Hybridization,    5′aagctgaagccgaatttccttgggataccagagga3′ (SEQ ID NO: 5).-   Rat keratin 19: Forward, 5′acagccagtacttcaagacc3′(SEQ ID NO: 6);    Reverse, 5′ctgtgtcagcacgcacgtta3′(SEQ ID NO: 7; Hybridization,    5′tggattccacaccaggcattgaccatgcca3′(SEQ ID NO: 8).-   Rat NCAM: Forward, 5′cagcgttggagagtccaaat3′(SEQ ID NO: 9); Reverse,    5′ttaaactcctgtggggttgg3′(SEQ ID NO: 10); Hybridization,    5′aaaccagcagcggatctcagtggtgtggaacgatgat3′(SEQ ID NO: 11).-   Rat IDX-1 Forward, 5′atcactggagcagggaagt3′(SEQ ID NO: 12) Reverse,    5′gctactacgtttcttatct3′ (SEQ ID NO: 13) Hybridization,    5′gcgtggaaaagccagtggg3′(SEQ ID NO: 14)-   Human nestin: Forward, 5′agaggggaattcctggag3′; (SEQ ID NO: 15)    Reverse, 5′ctgaggaccaggactctcta3′; (SEQ ID NO: 16) Hybridization,    5′tatgaacgggctggagcagtctgaggaaagt3′.(SEQ ID NO: 17)-   Human keratin: Forward, 5′cttttcgcgcgcccagcatt3′;(SEQ ID NO: 18)    Reverse, 5′gatcttcctgtccctcgagc3′;(SEQ ID NO: 19) Hybridization,    5′aaccatgaggaggaaatcagtacgctgagg3′.(SEQ ID NO: 20)-   Human glucagon: Forward, 5′atctggactccaggcgtgcc3′;(SEQ ID NO: 21)    Reverse, 5′agcaatgaattccttggcag3′; (SEQ ID NO: 22) Hybridization,    5′cacgatgaatttgagagacatgctgaaggg3′; (SEQ ID NO: 23)

Primers were selected from two different exons and encompassed at leastone intronic sequence. In addition, an RT minus control was run for mostsamples. PCR cycling was at 94° C. for 1 min followed by 94° C. for 10secs, 58/56° C. for 10 secs, 72° C. for 1 min, 35 cycles, and 72° C. for2 min. The annealing temperature was 58° C. for rat nestin and 56° C.for the remaining primer pairs.

For Southern hybridization oligonucleotide probes were radiolabeled withT4 polynucleotide kinase and γ³²P ATP. Radiolabeled probes werehybridized to PCR products that had been transferred to nylon membranesat 37° C. for one hour, then washed in 1×SSC+0.5% SDS at 55° C. for10-20 min or 0.5×SCC+0.5% SDS at 42° for the human PCR products.

The RT-PCR generated products of the correctly predicted size (FIG. 8E,upper panels) and were confirmed by Southern blotting (FIG. 8E, lowerpanels) and by DNA sequencing of the products. These data demonstratethe identification of a new cell type in pancreatic islets thatexpresses nestin and may represent an islet pluripotential stem cellsimilar to the nestin-positive stem cells in the central nervous system.

EXAMPLE 7

In vitro Proliferation of Nestin Positive Stem Cells

The ability of nestin-positive stem cells to proliferate in vitro wasdetermined.

Islets prepared from 60 day-old rats or a normal adult human were firstplated on concanavalin-A-coated dishes and cultured in modified RPMI1640 medium containing 10% fetal bovine serum for four days to purge theislet preparation of fibroblasts and other non-islet cells that adheredto the ConA-coated plates. The islets that did not adhere to the platesunder these culture conditions were collected and transferred to 12-wellplates (without ConA coating) containing the same modified RPMI 1640medium now additionally supplemented with bFGF and EGF (20 ng/mL each).The growth factors bFGF and EGF together were selected because they areknown to stimulate the proliferation of neural stem cells derived fromependyma of the brain (Reynolds and Weiss, 1996, Dev. Biol., 175:1-13).The islets attached to the plates and cells slowly grew out of the isletas a monolayer (estimated cell doubling time 40-45 hrs in human cells).The outgrowing monolayer of cells were phenotypically homogenous (FIG.9A, panel 1) and expressed nestin (FIG. 9A, panel 2). Rat cells werepicked from the monolayer (batches of at least 20-30 cells), subclonedinto 12-well plates, and incubated with the modified RPMI 1640 medium(11.1 mM glucose) containing bFGF and EGF. The subcloned cells grewrapidly and became confluent at six days with an estimated cell doublingtime of 12-15 hrs (FIG. 9A, panel 3), and by 12 days formed wave-likestructures. After 15-17 days of culture, the cells formed islet-likeclusters (ILCs) (FIG. 9A, panel 4). Similar cells were cloned from humanislets (FIG. 9B). Upon reaching confluence (FIG. 9B, panel 1), the humancells migrated to form large vacuolated structures in the dish (FIG. 9B,panels 2 and 3). The cells lining the large spaces then changedmorphology, rounded, and aggregated together forming three dimensionalILCs (FIG. 9B, panels 4-6).

Indicators of differentiation of these nestin-positive islet progenitorcells (NIPs) that formed these ILCs were characterized by RT-PCR andSouthern blot and found that they express the endocrine marker NCAM(neural cell adhesion molecule) (Cirulli et al., 1994, J. Cell Sci.,107:1429-36) (FIG. 9C, right panel) and the ductal cell marker CK19(cytokeratin 19) (Bouwens et al., 1998, J. Pathol., 184:234-9; Bouwenset al., 1995, J. Histochem. Cytochem., 43:245-53; Bouwens et al., 1994,Diabetes, 43:1279-93) (FIG. 9C, left panels). At this stage of thestudies it was concluded that when the NIPs became confluent andaggregated into islet-like cell clusters, they began to expresspancreatic genes (NCAM and CK19), but were limited in expression ofislet genes because of the absence of growth factors essential for theirdifferentiation to endocrine cells. It was also recognized that thedifferentiation of a progenitor cell population typically requires firsta proliferative phase and then quiescence of proliferation in thepresence of differentiation-specific morphogen growth factors. Thereforethe culture conditions were modified in some instances by replacing themedia containing 11.1 mM glucose, bFGF and EGF, which inducesproliferation of cells, with media containing lower glucose (2.5 mM),which is less proliferative, and the factors HGF/Scatter Factor orbetacellulin and Activin A. Glucose is a known proliferative factor forpancreatic islet β-cells (Swenne, 1992, Diabetologia, 35:193-201;Bonner-Weir, 1989, Diabetes, 38:49-53) and both HGF/Scatter Factor andActivin A have been shown to differentiate the pancreatic ductal cellline AR42J into an endocrine phenotype that produces insulin, glucagon,and other pancreatic endocrine cell proteins (Mashima et al., 1996,Endocrinology, 137:3969-76; Mashima et al., 1996, J. Clin. Invest.,97:1647-54).

Cultures containing ILCs expressed the pancreas-specific homeodomainprotein IDX-1 by immunocytochemistry (FIG. 10A, upper panel), RT-PCR andSouthern blot (FIG. 10B), and by Western immunoblot (FIG. 10C). The ILCsalso expressed the mRNA encoding proglucagon as seen by RT-PCR (FIG.10D) and produced immunoreactive glucagon, glucagon-like peptide-1, andinsulin. Radioimmunoassays of media obtained following 72-96 h ofculture of islet-like clusters in several wells gave values of 40-80pg/ml GLP-1, 30-70 pg/ml glucagon, 29-44 pg/ml insulin.Radioimmunoassays were performed as follows.

Insulin and glucagon concentrations in culture media were determined byultra sensitive radioimmunoassay kits purchased from Linco Research Inc.and DPC Inc., respectively. The antisera supplied in the respective kitsare guinea pig anti-human insulin and rabbit anti-human glucagon. GLP-1secretion was measured with an anti-human GLP-1(7-36)amide rabbitpolyclonal antiserum raised by immunization of a rabbit with a syntheticpeptide CFIAWLVKGR (SEQ ID NO: 54) amide conjugated to keyhole limpethemocyanin. The antiserum is highly specific for the detection ofGLP-1(7-36)amide and only weakly detects proglucagon. The sensitivitylevels for these assays are 6 pg/mL, 13 pg/mL and 10.2 pg/mL,respectively.

Incubation of the ILCs for 7 days in 10 mM nicotinamide, as described byRamiya et al. (Ramiya et al., 2000, Nat. Med., 6:278-282), increasedinsulin secretion by 2- to 3-fold.

Several additional pancreatic markers were expressed in differentiatedNIPs such as glucose transporter-2 (Wang et al., 1998), synaptophysin,and HGF (Menke et al., 1999) as shown in FIG. 15. To determine whetherthe differentiating NIPs may have properties of pancreatic exocrinetissue, we used RT-PCR and detected the expression of amylase andprocarboxypeptidase (FIG. 15).

Some cultures of NIPs containing stem cells also expressed the mRNAencoding proglucagon and insulin as seen by RT-PCR (FIGS. 16A and B).

The expression of IDX-1 is of particular importance because it isrecognized to be a master regulator of pancreas development, andparticularly to be required for the maturation and functions of thepancreatic islet β-cells that produce insulin (Stoffers et al., 1997,Trends Endocrinol. Metab., 8:145-151).

Because the neogenesis of new islets is also known to occur bydifferentiation of cells in pancreatic ducts, particularly during theneonatal period (rats and mice) but to some extent throughout adult life(Bonner-weir et al., 1993, Diabetes, 42:1715-1720; Rosenberg, 1995, CellTransplant, 4:371-383; Bouwens et al., 1996, Virchows Arch.,427:553-560), nestin expression was analyzed in the pancreatic ducts ofadult rats. By dual fluorescence immunocytochemistry with antisera tonestin and to cytokeratin 19, a marker of ductal epithelium, nestin isstrongly expressed in localized regions of both the large and smallducts, as well as in some centrolobular ducts within the exocrine acinartissue (FIGS. 11A and 11B). Remarkably, the localized regions of nestinexpression in the ducts are mostly devoid of staining with the anti CK19antiserum. Further, the nestin-positive cells in the ducts appear tohave a morphology that is distinct from that of the epithelial cells.The epithelial cells consist of a homogenous population of cuboidal,rounded cells, whereas the nestin-positive cells are nucleated,serpiginous and appear to reside in the interstices among or aroundepithelial cells (FIG. 11C).

Thus, CK19 is not expressed in the majority of ductal cells that expressnestin suggesting that these nestin-expressing cells located within thepancreatic ducts are a passenger population of cells distinct from theductal epithelial cells and are stem cells that have not yetdifferentiated into a ductal or endocrine phenotype. The finding oflocalized populations of nestin-expressing cells within the pancreaticducts and islets of the adult rat pancreas further supports the ideathat rat pancreatic ducts contain cells that are progenitors of isletcells (neogenesis), but these progenitors are not a subpopulation ofductal epithelial cells per se.

EXAMPLE 8

Transplantation of Pancreatic Stem Cells Engineered to Express IDX-1 inHuman Subjects with Diabetes Mellitus

Islets isolated from pig or human donor pancreata, or from pancreaticbiopsy of eventual human transplant recipient are cultured ex vivo inconditions that stimulate outgrowth of stem cells. Stem cells are thenisolated away from islets (cloned), expanded in vitro in proliferationmedia containing bFGF-2, EGF, and 11.1 mM glucose, transfected/injectedwith an expression vector containing DNA encoding transcription factorIDX-1, and transplanted into a diabetic recipient. Alternatively,IDX-1-transfected stem cells are treated with GLP-1, or otherdifferentiation morphogens or growth factors for 1-3 days beforetransplantation to initiate processes of differentiation of engineeredstem cells to β-cells. In one embodiment, stem cells are neitherexpanded or differentiated prior to administration to the recipient orare only expanded or differentiated prior to administration to therecipient. In one embodiment, GLP-1 is administered to the recipientduring and for several days after transplantation to stimulatedifferentiation of stem cells and encourage successful engraftment.According to this method, xenographs (pig islets) or allographs (humanislets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are carried out. It ishypothesized that when transplanted to a host recipient the stem cellgenetic repertoire is reprogrammed so that the host recognizes the stemcells (in the case of xenographs or allographs) as self, such thatimmune intolerance and graft rejection and destruction by autoimmunity(type 1 diabetes) does not occur.

EXAMPLE 9

Transplantation of Pancreatic Stem Cells Cultured to StimulateExpression of IDX-1 in Human Subjects with Diabetes Mellitus

Islets isolated as described are cultured ex vivo for several days inconditions that stimulate first the expansion (proliferation) of stemcells that exist within the islets and then the expression oftranscription factor IDX-1. The proliferation of stem cells is achievedby culturing the islets in media containing bFGF-2, EGF, and 11.1 mMglucose. Induction of the expression of IDX-1 is achieved by incubationin the presence of GLP-1 and 2 mM glucose. The islets so preconditionedby the treatments described are transplanted to the host recipient.Additionally, the host recipient may be administered GLP-1 during andfor several days after the transplantation to further expand anddifferentiate stem cells to insulin-producing cells to enhance successof engraftment.

According to this method, xenographs (pig islets), allographs (humanislets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are effectuated.

EXAMPLE 10

Xenogeneic Transplantation of Pancreatic Stem Cells into the Kidney

Human nestin-positive-islet progenitor cells (NIPS) were isolated asdescribed, and transplanted under the renal capsules of eight C57B16mice that were not immunosuppressed. The transplanted human cells werenot rejected by the mouse recipient. Current understanding is that axenograft, such as human tissue, would be rejected by the mouse within5-10 days. Contrary to current understanding, we found that in 8 of the8 non-immunosuppressed mice tested to date, all of the transplantssuccessfully engrafted and proliferated into large masses of tissueengulfing the pole of the kidney by one month (30-38 days) after atransplantation of approximately 10⁵ to 10⁶ cells.

One C57B16 mouse was sacrificed and determined to have a large area ofnew growth at the site of transplantation. A section of the kidney thatincluded the new tissue was divided into two pieces; one piece wasfrozen for frozen section histology, and the other piece was fixed inparaformaldehyde for paraffin section histology. Frozen sections wereprepared and stained with hematoxylin and eosin (H&E) and antisera tovarious islet cell antigens.

Examination of the H&E stained kidney section demonstrated the presenceof a new growth that was not part of the kidney, exhibiting apleiomorphic morphology consisting of a mixed mesenchymal and epithelialtissue containing hepatic, neural, ductal, adipodipic and hematopoeticcomponents. Photomicrographs of the kidney section demonstrated that thenew growth seemed to be invading the renal parenchyme, and theglomeruli. Specific immunostaining with human-specific (not mouse)antisera revealed cords of immunopositive cells staining forhuman-specific keratins, vimentin, and the CD45 leukocyte antigenspecific for human hematapoetic lymphocytes. The kidney of a secondC57B16 mouse also had a similar looking new growth at the site of theNIP transplantation.

The paraffin section of the NIP-engrafted kidney of a C57B16 mouse (thefirst mouse to be sacrificed) was examined. The tissue block that wasexamined was from the top of the kidney and showed the foreign tissue tobe well contained under the renal capsule with no signs of “invasion”into the renal parenchyma. Notably, amongst the pleiomorphic-lookinggraft tissues were areas that resembled renal parenchyma. Without beingbound to theory one hypothesis is that the graft consists of stem cellstrying to differentiate and that the stem cells are not “invading” butsimply migrating and proliferating and looking for a niche, i.e.mesenchymal instructions. They may be receiving cues from the kidney andmay be attempting to differentiate into kidney. The graft cells may notbe malignant, but may be just stem cells attempting to carry out theirfunction.

EXAMPLE 11

Xenogeneic Transplantation of Pancreatic Stem Cells into the Pancreas

Human nestin-positive-islet progenitor cells (NIPS) are isolated asdescribed, and transplanted into the pancreas of mice that are notimmunosuppressed and are (a) injured by streptozotocin (to producestreptozotocin induced diabetes) treatment or (b) NOD mice in whichthere is an ongoing islet is with inflammation.

The pancreas of the transplanted animals is examined to determine if theNIPs find their proper niche, receive instructions from the isletregion, and differentiate into islet (β-cell) cells.

EXAMPLE 12

Treatment of Diabetes by Xenogeneic Transplantation of Pancreatic StemCells

Human islets are isolated as described and cultured for several days invitro to expand the stem cell population. Human NIPS are transplanted tothe liver via the portal vein (according to conventional procedures wellknown in the art for transplantation to the liver.

Alternatively, a population of human NIPs (isolated as described) areintroduced into the blood stream. In certain embodiments, the human NIPSare introduced via the pancreatic artery, to direct them to the diabeticpancreas.

A population of control (untransplanted animals) and transplantedanimals are analyzed for amelioration of the symptoms of diabetes (e.g.blood glucose levels, insulin levels, number of pancreatic β-cells.

EXAMPLE 13

Identification of Nestin Positive Stem Cells in the Liver

Rat livers were isolated and frozen section were prepared according tomethods known in the art and described herein.

Frozen sections of rat liver (6 μM) were immunostained with a rabbitpolyclonal anti-nestin serum. The immunofluorescent signal was developedby reaction of anti-donkey IgG serum tagged with the fluorophore, Cy3(yellow-orange color. Nestin-positive cells surrounding a possible largebiliary duct are depicted in FIG. 13A. Clusters of nestin positive cellssurrounding several small biliary ducts are depicted in FIG. 13B.

EXAMPLE 14

Differentiation of NIPs Toward Hepatic Phenotype

Because of the reported apparent commonalties between hepatic stem cells(oval cells), hepatic stellate cells, and progenitor cells in thepancreas, and the observations that following some injuries, theregenerating pancreas undergoes liver metaplasia (Slack, 1995; Reddy etal., 1991; Bisgaard et al., 1991; Rao et al., 1996), we performed RT-PCRto detect liver-expressed genes in the stem cells. PCR products wereobtained for XBP-1, a transcription factor required for hepatocytedevelopment (Reimold et al., 2000), and transthyretin, a liver acutephase protein. Several other liver markers were also expressed such asα-fetoprotein (Dabeva et al., 2000), E-Cadherin (Stamatoglou et al.,1992), c-MET (Ikeda et al., 1998), HGF (Skrtic et al., 1999) andsynaptophysin (Wang et al., 1998); see FIG. 15)) The expression ofproteins shared by the pancreas and liver, such as HGF andsynaptophysin, may reflect their common origin from the embryonicforegut endoderm, and represent differentiation toward either pancreaticor hepatic phenotypes.

References

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Other Embodiments

Other Embodiments are within the claims that follow.

1. A method of treating a patient with diabetes mellitus, comprising thesteps of: (a) isolating a nestin-positive pancreatic stem cell from apancreatic islet of a donor; (b) expanding the stem cell to produce aprogenitor cell; (c) differentiating the progenitor cell in culture toform pseudo-islet like aggregates; and (d) transferring the pseudo-isletlike aggregates into the patient, wherein the patient does not serve asthe donor for said stem cells of step (a), and wherein said transferringstep (d) treats diabetes mellitus.
 2. The method of claim 1, wherein thepatient is a human and the donor for said stem cells of step (a) is anon-human mammal.
 3. The method of claim 1 or 2, wherein the patient isnot treated with an immunosuppressive agent prior to step (b).
 4. Themethod of claim 1, wherein the step of expanding is performed in thepresence of an agent selected from the group consisting of EpidermanGrowth Factor (EGF), basic Fibroblast Growth Factor-2 (bFGF-2), highglucose, Keratinocyte Growth Factor KGF, Hepatocyte GrowthFactor/Scatter Factor (HGF/SF), Glucagon-like-Peptide-1 (GLP-1),exendin-4, Islet/Duodenum Homeobox-1 (IDX-1), a nucleic acid moleculeencoding Islet/Duodenum Homeobox-1 (IDX-1), betacellulin, activin A,Transforming Growth Factor-β (TGF-β), and combinations thereof.
 5. Themethod of claim 1, wherein the step of transferring is performed viaendoscopic retrograde injection.
 6. The method of claim 1 additionallycomprising the step of: (e) treating the patient with animmunosuppressive agent.
 7. The method of claim 6 wherein saidimmunosuppressive agent prevents an immune response.
 8. The method ofclaim 6 wherein said immunosuppressive agent delays the occurrence of animmune response.
 9. The method of claim 6 wherein said immunosuppressiveagent decreases the intensity of an immune response.
 10. The method ofclaim 6, 7, 8 or 9 wherein the immune response is transplant rejection.11. The method of claim 6, wherein the immunosuppressive agent isselected from the group consisting of FK-506, cyclosporin, and GAD65antibodies.
 12. A method of transplanting into a mammal, comprising thesteps of: (a) isolating a nestin-positive pancreatic stem cell from apancreatic islet of a donor; (b) expanding the stem cell to produce aprogenitor cell; (c) differentiating the progenitor cell in culture toform pseudo-islet like aggregates; and (d) transferring the pseudo-isletlike aggregates into the mammal.
 13. The method of claim 12, wherein themammal serves as the donor for said stem cells of step (a).
 14. Themethod of claim 12, wherein the mammal does not serve as the donor forsaid stem cells of step (a).
 15. The method of claim 12, wherein themammal is a human and the donor for said stem cells of step a is anon-human mammal.
 16. The method of claim 14 or 15, wherein the mammalis not treated with an immunosuppressive agent prior to step (b). 17.The method of claim 12, wherein the step of expanding is performed inthe presence of an agent selected from the group consisting of EpidermalGrowth Factor (EGF), basic Fibroblast Growth Factor-2 (bFGF-2), highglucose, Keratinocyte Growth Factor (KGF), Hepatocyte GrowthFactor/Scatter Factor (HGF/SF), Glucagon-like-Peptide-1 (GLP-1),exendin-4, Islet/Duodenum Homeobox-1 (IDX-1), a nucleic acid moleculeencoding Islet/Duodenum Homeobox-1 (IDX-1), betacellulin, activin A,Transforming Growth Factor-β (TGF-β), and combinations thereof.
 18. Themethod of claim 12, wherein the step of transferring is performed viaendoscopic retrograde injection.
 19. The method of claim 12 additionallycomprising the step of: (e) treating the mammal with animmunosuppressive agent.
 20. The method of claim 19 wherein saidimmunosuppressive agent prevents an immune response.
 21. The method ofclaim 19 wherein said immunosuppressive agent delays the occurrence ofan immune response.
 22. The method of claim 19 wherein saidimmunosuppressive agent decreases the intensity of an immune response.23. The method of claim 20, 21 or 22 wherein the immune response istransplant rejection.
 24. The method of claim 19, wherein theimmunosuppressive agent is selected from the group consisting of FK-506,cyclosporin, and GAD65 antibodies.
 25. A method of treating a subjectwith diabetes mellitus, comprising the steps of: (a) isolating anestin-positive pancreatic stem cell from a pancreatic islet of a humandonor; (b) expanding the stem cell to produce a progenitor cell; (c)differentiating the progenitor cell in culture to form pseudo-islet likeaggregates; and (d) transferring the pseudo-islet like aggregates intosaid subject, wherein said subject does not serve as the donor for saidstem cells of step (a), and wherein said transferring step (d) treatsdiabetes mellitus.
 26. The method of claim 25, wherein said subject isnot treated with an immunosuppresive agent prior to step (b).
 27. Themethod of claim 25, wherein the step of expanding is performed in thepresence of an agent selected from the group consisting of EpidermalGrowth Factor (EGF), basic Fibroblast Growth Factor-2 (bFGF-2), highglucose, Keratinocyte Growth Factor (KGF), Hepatocyte GrowthFactor/Scatter Factor (HGF/SF), Glucagon-like-Peptide-1 (GLP-1),exendin-4, Islet/Duodenum Homeobox-1 (IDX-1), a nucleic acid moleculeencoding Islet/Duodenum Homeobox-1 (IDX-1), betacellulin, activin A,Transforming Growth Factor-β (TGF-β), and combinations thereof.
 28. Themethod of claim 25, wherein the step of transferring is performed viaendoscopic retrograde injection.
 29. The method of claim 25 additionallycomprising the step of: (e) treating said subject with animmunosuppressive agent.
 30. The method of claim 29 wherein saidimmunosuppressive agent prevents an immune response.
 31. The method ofclaim 29 wherein said immunosuppressive agent delays the occurrence ofan immune response.
 32. The method of claim 29 wherein saidimmunosuppressive agent decreases the intensity of an immune response.33. The method of claim 30, 31 or 32 wherein the immune response istransplant rejection.
 34. The method of claim 29, wherein theimmunosuppressive agent is selected from the group consisting of FK-506,cyclosporin, and GAD65 antibodies.
 35. A method of transplanting into amammal, comprising the steps of: (a) isolating a nestin-positivepancreatic stem cell from a pancreatic islet of a human donor; (b)expanding the stem cell to produce a progenitor cell; (c)differentiating the progenitor cell in culture to form pseudo-islet likeaggregates; and (d) transferring the pseudo-islet like aggregates intothe mammal.
 36. The method of claim 35, wherein the mammal serves as thedonor for said stem cells of step (a).
 37. The method of claim 35,wherein the mammal does not serve as the donor for said stem cells ofstep (a).
 38. The method of claim 37, wherein the mammal is not treatedwith an immunosuppressive agent prior to step (b).
 39. The method ofclaim 35, wherein the step of expanding is performed in the presence ofan agent selected from the group consisting of Epidermal Growth Factor(EGF), basic Fibroblast Growth Factor-2 (bFGF-2), high glucose,Keratinocyte Growth Factor (KGF), Hepatocyte Growth Factor/ScatterFactor (HGF/SF), Glucagon-like-Peptide-1 (GLP-1), exendin-4,Islet/Duodenum Homeobox-1 (IDX-1), a nucleic acid molecule encodingIslet/Duodenum Homeobox-1 (IDX-1), betacellulin, activin A, TransformingGrowth Factor-β (TGF-β), and combinations thereof.
 40. The method ofclaim 35, wherein the step of transferring is perform via endoscopicretrograde injection.
 41. The method of claim 35 additionally comprisingthe step of: (e) treating the mammal with an immunosuppresive agent. 42.The method of claim 41 wherein said immunosuppressive agent prevents animmune response.
 43. The method of claim 41 wherein saidimmunosuppressive agent delays the occurrence of an immune response. 44.The method of claim 41 wherein said immunosuppressive agent decreasesthe intensity of an immune response.
 45. The method of claim 42, 43, or44, wherein the immune response is transplant rejection.
 46. The methodof claim 41, wherein the immunosuppressive agent is selected from thegroup consisting of FK-506, cyclosporin, and GAD65 antibodies.